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1.
Appl Biochem Biotechnol ; 171(4): 954-62, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23912211

RESUMO

A key enzyme for the biosynthesis and bioengineering of heparin, 3-O-sulfotransferase-1 (3-OST-1), was expressed and purified in Gram-positive Bacillus subtilis and Bacillus megaterium. Western blotting, protein sequence analysis, and enzyme activity measurement confirmed the expression. The enzymatic activity of 3-OST-1 expressed in Bacillus species were found to be similar to those found when expressed in Escherichia coli. The endotoxin level in 3-OST-1 from B. subtilis and B. megaterium were 10(4)-10(5)-fold lower than that of the E. coli-expressed 3-OST-1, which makes the Bacillus expression system of particular interest for the generation of pharmaceutical grade raw heparin from nonanimal sources.


Assuntos
Bacillus megaterium/enzimologia , Bacillus subtilis/enzimologia , Sulfotransferases/metabolismo , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo
2.
Electrophoresis ; 33(11): 1531-7, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22736353

RESUMO

The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment.


Assuntos
Sulfatos de Condroitina/análise , Eletroforese/métodos , Heparina/análise , Heparina/normas , Sulfatos de Condroitina/química , Contaminação de Medicamentos , Heparina/química
3.
Anal Bioanal Chem ; 403(6): 1491-500, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22526635

RESUMO

Sulfotransferases are enzymes that catalyze the transfer of sulfo groups from a donor, for example 3'-phosphoadenosine 5'-phosphosulfate, to an acceptor, for example the amino or hydroxyl groups of a small molecule, xenobiotic, carbohydrate, or peptide. These enzymes are important targets in the design of novel therapeutics for treatment of a variety of diseases. This review examines assays used for this important class of enzyme, paying particular attention to sulfotransferases acting on carbohydrates and peptides and the major challenges associated with their analysis.


Assuntos
Sulfotransferases/metabolismo , Fluorometria , Espectrometria de Massas , Radiometria
4.
Biotechnol Appl Biochem ; 59(6): 420-8, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23586950

RESUMO

Heparin is a widely used clinical anticoagulant that is prepared from pig intestine. A contamination of heparin in 2008 has led to a reexamination of animal-derived pharmaceuticals. A bioengineered heparin prepared by bacterial fermentation and chemical and enzymatic processing is currently under development. This study examines the challenges of reducing or removing endotoxins associated with this process that are necessary to proceed with preclinical in vivo evaluation of bioengineered heparin. The current process is assessed for endotoxin levels, and strategies are examined for endotoxin removal from polysaccharides and enzymes involved in this process.


Assuntos
Bioengenharia/métodos , Contaminação de Medicamentos/prevenção & controle , Endotoxinas/análise , Endotoxinas/isolamento & purificação , Heparina/genética , Heparina/metabolismo , Cromatografia , Endotoxinas/química , Etanolaminas/química , Heparina/biossíntese , Interações Hidrofóbicas e Hidrofílicas , Oxirredução , Polimixina B/química
5.
J Pharm Sci ; 98(11): 4017-26, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19642166

RESUMO

In 2008, heparin (active pharmaceutical ingredient, API) lots were associated with anaphylactoid-type reactions. Oversulfated chondroitin sulfate (OSCS), a semi-synthetic glycosaminoglycan (GAG), was identified as a contaminant and dermatan sulfate (DS) as an impurity. While DS has no known toxicity, OSCS was toxic leading to patient deaths. Heparins, prepared before these adverse reactions, needed to be screened for impurities and contaminants. Heparins were analyzed using high-field (1)H-NMR spectroscopy. Heparinoids were mixed with a pure heparin and analyzed by (1)H-NMR to assess the utility of (1)H-NMR for screening heparin adulterants. Sensitivity of heparinoids to deaminative cleavage, a method widely used to depolymerize heparin, was evaluated with polyacrylamide gel electrophoresis to detect impurities and contaminants, giving limits of detection (LOD) ranging from 0.1% to 5%. Most pharmaceutical heparins prepared between 1941 and 2008 showed no impurities or contaminants. Some contained DS, CS, and sodium acetate impurities. Heparin prepared in 2008 contained OSCS contaminant. Heparin adulterated with heparinoids showed additional peaks in their high-field (1)H-NMR spectra, clearly supporting NMR for monitoring of heparin API with an LOD of 0.5-10%. Most of these heparinoids were stable to nitrous acid treatment suggesting its utility for evaluating impurities and contaminants in heparin API.


Assuntos
Anticoagulantes/análise , Contaminação de Medicamentos , Eletroforese/métodos , Heparina/análise , Espectroscopia de Ressonância Magnética/métodos , Anticoagulantes/química , Sulfatos de Condroitina/análise , Dermatan Sulfato/análise , Heparina/química , Heparinoides/análise , Humanos , Estrutura Molecular , Ácido Nitroso/análise , Sensibilidade e Especificidade
6.
Carbohydr Res ; 344(10): 1190-6, 2009 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-19476923

RESUMO

The novel low-molecular-weight chitosan polysulfate (MW 5120-26,200 Da) was prepared using the depolymerization of chitosan with papain (EC. 3.4.22.2). The sulfonation of depolymerized products was performed using chlorosulfonic acid in N,N-dimethylformamide under semi-heterogeneous conditions. The structures of the products were characterized by FTIR, (13)C NMR, and (1)H NMR (1D, 2D NMR) spectroscopy. The present study sheds light on the mechanism of anticoagulant activity of chitosan polysulfate. Anticoagulant activity was investigated by an activated partial thromboplastin assay, a thrombin time assay, a prothrombin time assay, and thrombelastography. Surface plasmon resonance also provided valuable data for understanding the relationship between the molecular binding of sulfated chitosan to two important blood clotting regulators, antithrombin III and heparin cofactor II. These results show that the principal mechanism by which this chitosan polysulfate exhibits anticoagulant activity is mediated through heparin cofactor II and is dependent on polysaccharide molecular weight.


Assuntos
Anticoagulantes/química , Anticoagulantes/farmacologia , Quitosana/química , Quitosana/farmacologia , Papaína/metabolismo , Ácidos Sulfônicos/química , Anticoagulantes/metabolismo , Antitrombina III/metabolismo , Quitosana/metabolismo , Cofator II da Heparina/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Peso Molecular
7.
Biochem Pharmacol ; 78(3): 292-300, 2009 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-19389385

RESUMO

An oversulfated chondroitin sulfate (OSCS) was identified as a contaminant to pharmaceutical heparin and severe anaphylactoid reactions were ascribed to this contaminant. An examination of the biochemistry underlying both the anticoagulant activity and the toxic effects of oversulfated chondroitin sulfate was undertaken. This study demonstrates that the anticoagulant activity of this oversulfated chondroitin sulfate is primarily dependent on heparin cofactor II mediated inhibition of thrombin. Heparin and oversulfated chondroitin sulfate binding to coagulation, kinin-kallikrein and complement proteins were studied by surface plasmon resonance. While oversulfated chondroitin sulfate binds tightly to antithrombin III, unlike heparin, OSCS does not induce antithrombin III to undergo the conformational change required for its inactivation of thrombin and factor Xa. In contrast to heparin, oversulfated chondroitin sulfate tightly binds factor XIIa suggesting a biochemical mechanism for the factor XIIa-based enhancement of vasoactive bradykinin production.


Assuntos
Anticoagulantes/efeitos adversos , Peptídeos Catiônicos Antimicrobianos/química , Proteínas Sanguíneas/química , Proteínas de Transporte/química , Sulfatos de Condroitina/química , Heparina/efeitos adversos , Sequência de Carboidratos , Contaminação de Medicamentos , Heparina/química , Cinética , Espectroscopia de Ressonância Magnética , Ressonância de Plasmônio de Superfície
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